Interacting Regions in Stat3 and c-Jun That Participate in Cooperative Transcriptional Activation

Abstract

Independent but closely spaced DNA binding sites for Stat3 and c-Jun are required for maximal enhancer function in a number of genes, including the gene encoding the interleukin-6 (IL-6)-induced acute-phase response protein, α₂-macroglobulin. In addition, a physical interaction of Stat3 with c-Jun, based on yeast two-hybrid interaction experiments, has been reported. Here we confirm the existence of an interaction between Stat3 and c-Jun both in vitro, with recombinant proteins, and in vivo, during transient transfection. Using fragments of both proteins, we mapped the interactive sites to the C-terminal region of c-Jun and to two regions in Stat3, within the coiled-coil domain and in a portion of the DNA binding domain distant from DNA contact sites. In transient-transfection experiments with the α₂-macroglobulin enhancer, Stat3 and c-Jun cooperated to yield maximal enhancer function. Point mutations of Stat3 within the interacting domains blocked both physical interaction of Stat3 with c-Jun and their cooperation in IL-6-induced transcription directed by the α₂-macroglobulin enhancer. While the amino acid sequences and the three-dimensional structures of Stat3 and Stat1 cores are very similar, fragments of Stat1 failed to bind c-Jun in vitro. Although Stat1 binds in vitro to the gamma interferon gene response (GAS) element in the α₂-macroglobulin enhancer, Stat1 did not stimulate transcription, nor did Stat1 and c-Jun cooperate in driving transcription controlled by the α₂-macroglobulin enhancer.

ACKNOWLEDGMENTS

We thank Lois Cousseau for preparing the manuscript; Jacqueline F. Bromberg, Daniel Besser, and Jillian J. Zhang for scientific discussions; and Yanxiang Zhao, David Jeruzalmi, and Xiaomin Chen for preparing the ribbon diagram of the Stat1 molecule.

This work was supported by NIH grants AI32489 and AI34420 to J.E.D. X.Z. is supported by NIH training grant CA09673. M.H.W. is supported by a Cancer Research Institute postdoctoral fellowship.